ABSTRACT
Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome.
Subject(s)
Benzamides/metabolism , Bridged Bicyclo Compounds/pharmacology , Heptanes/pharmacology , Lysosomes/drug effects , Vesicular Transport Proteins/metabolism , Activating Transcription Factor 6/metabolism , Animals , Benzamides/chemistry , Benzamides/pharmacology , Bridged Bicyclo Compounds/therapeutic use , Epithelial Cells/cytology , Epithelial Cells/metabolism , Female , Frameshift Mutation , Heptanes/therapeutic use , Humans , Imidazoline Receptors/antagonists & inhibitors , Imidazoline Receptors/genetics , Imidazoline Receptors/metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Kidney/cytology , Kidney/metabolism , Kidney/pathology , Kidney Diseases/metabolism , Kidney Diseases/pathology , Lysosomes/metabolism , Male , Mice , Mice, Transgenic , Mucin-1/chemistry , Mucin-1/genetics , Mucin-1/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Unfolded Protein Response/drug effects , Vesicular Transport Proteins/chemistryABSTRACT
Siglec-9 is a sialic-acid-binding lectin expressed predominantly on myeloid cells. Aberrant glycosylation occurs in essentially all types of cancers and results in increased sialylation. Thus, when the mucin MUC1 is expressed on cancer cells, it is decorated by multiple short, sialylated O-linked glycans (MUC1-ST). Here we found that this cancer-specific MUC1 glycoform, through engagement of Siglec-9, 'educated' myeloid cells to release factors associated with determination of the tumor microenvironment and disease progression. Moreover, MUC1-ST induced macrophages to display a tumor-associated macrophage (TAM)-like phenotype, with increased expression of the checkpoint ligand PD-L1. Binding of MUC1-ST to Siglec-9 did not activate the phosphatases SHP-1 or SHP-2 but, unexpectedly, induced calcium flux that led to activation of the kinases MEK-ERK. This work defines a critical role for aberrantly glycosylated MUC1 and identifies an activating pathway that follows engagement of Siglec-9.
Subject(s)
Antigens, CD/metabolism , Mucin-1/metabolism , Neoplasms/immunology , Neoplasms/metabolism , Sialic Acid Binding Immunoglobulin-like Lectins/metabolism , Tumor Microenvironment/immunology , Antigens, CD/genetics , Biomarkers , Cell Differentiation , Cell Line , Dendritic Cells/immunology , Dendritic Cells/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression , Glycosylation , Humans , Macrophages/immunology , Macrophages/metabolism , Mitogen-Activated Protein Kinases/metabolism , Myeloid Cells/cytology , Myeloid Cells/immunology , Myeloid Cells/metabolism , Neoplasms/genetics , Neoplasms/pathology , Phenotype , Protein Binding , Sialic Acid Binding Immunoglobulin-like Lectins/geneticsABSTRACT
Brain metastatic breast cancer is particularly lethal largely due to therapeutic resistance. Almost half of the patients with metastatic HER2-positive breast cancer develop brain metastases, representing a major clinical challenge. We previously described that cancer-associated fibroblasts are an important source of resistance in primary tumors. Here, we report that breast cancer brain metastasis stromal cell interactions in 3D cocultures induce therapeutic resistance to HER2-targeting agents, particularly to the small molecule inhibitor of HER2/EGFR neratinib. We investigated the underlying mechanisms using a synthetic Notch reporter system enabling the sorting of cancer cells that directly interact with stromal cells. We identified mucins and bulky glycoprotein synthesis as top-up-regulated genes and pathways by comparing the gene expression and chromatin profiles of stroma-contact and no-contact cancer cells before and after neratinib treatment. Glycoprotein gene signatures were also enriched in human brain metastases compared to primary tumors. We confirmed increased glycocalyx surrounding cocultures by immunofluorescence and showed that mucinase treatment increased sensitivity to neratinib by enabling a more efficient inhibition of EGFR/HER2 signaling in cancer cells. Overexpression of truncated MUC1 lacking the intracellular domain as a model of increased glycocalyx-induced resistance to neratinib both in cell culture and in experimental brain metastases in immunodeficient mice. Our results highlight the importance of glycoproteins as a resistance mechanism to HER2-targeting therapies in breast cancer brain metastases.
Subject(s)
Brain Neoplasms , Breast Neoplasms , Drug Resistance, Neoplasm , Glycocalyx , Quinolines , Receptor, ErbB-2 , Stromal Cells , Humans , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Female , Brain Neoplasms/secondary , Brain Neoplasms/metabolism , Brain Neoplasms/drug therapy , Brain Neoplasms/pathology , Brain Neoplasms/genetics , Receptor, ErbB-2/metabolism , Receptor, ErbB-2/genetics , Glycocalyx/metabolism , Animals , Cell Line, Tumor , Stromal Cells/metabolism , Stromal Cells/pathology , Quinolines/pharmacology , Mice , Cell Communication , Coculture Techniques , Mucin-1/metabolism , Mucin-1/genetics , Signal Transduction , ErbB Receptors/metabolism , ErbB Receptors/antagonists & inhibitorsABSTRACT
Tibroviruses are novel rhabdoviruses detected in humans, cattle, and arthropods. Four tibroviruses are known to infect humans: Bas-Congo virus (BASV), Ekpoma virus 1 (EKV-1), Ekpoma virus 2, and Mundri virus. However, since none of them has been isolated, their biological properties are largely unknown. We aimed to characterize the human tibrovirus glycoprotein (G), which likely plays a pivotal role in viral tropism and pathogenicity. Human tibrovirus Gs were found to share some primary structures and display 14 conserved cysteine residues, although their overall amino acid homology was low (29%-48%). Multiple potential glycosylation sites were found on the G molecules, and endoglycosidase H- and peptide-N-glycosidase F-sensitive glycosylation was confirmed. AlphaFold-predicted three-dimensional (3D) structures of human tibrovirus Gs were overall similar. Membrane fusion mediated by these tibrovirus Gs was induced by acidic pH. The low pH-induced conformational change that triggers fusion was reversible. Virus-like particles (VLPs) were produced by transient expression of Gs in cultured cells and used to produce mouse antisera. Using vesicular stomatitis Indiana virus pseudotyped with Gs, we found that the antisera to the respective tibrovirus VLPs showed limited cross-neutralizing activity. It was also found that human C-type lectins and T-cell immunoglobulin mucin 1 acted as attachment factors for G-mediated entry into cells. Interestingly, BASV-G showed the highest ability to utilize these molecules. The viruses infected a wide range of cell lines with preferential tropism for human-derived cells whereas the preference of EKV-1 was unique compared with the other human tibroviruses. These findings provide fundamental information to understand the biological properties of the human tibroviruses. IMPORTANCE: Human tibroviruses are poorly characterized emerging rhabdoviruses associated with either asymptomatic infection or severe disease with a case fatality rate as high as 60% in humans. However, the extent and burden of human infection as well as factors behind differences in infection outcomes are largely unknown. In this study, we characterized human tibrovirus glycoproteins, which play a key role in virus-host interactions, mainly focusing on their structural and antigenic differences and cellular tropism. Our results provide critical information for understanding the biological properties of these novel viruses and for developing appropriate preparedness interventions such as diagnostic tools, vaccines, and effective therapies.
Subject(s)
Viral Envelope Proteins , Humans , Animals , Viral Envelope Proteins/metabolism , Viral Envelope Proteins/genetics , Mice , Glycosylation , Virus Internalization , Viral Tropism , Cell Line , Mucin-1/metabolism , HEK293 Cells , Antibodies, Viral/immunology , Amino Acid SequenceABSTRACT
Polymorphism of the gene encoding mucin 1 (MUC1) is associated with skeletal and dental phenotypes in human genomic studies. Animals lacking MUC1 exhibit mild reduction in bone density. These phenotypes could be a consequence of modulation of bodily Ca homeostasis by MUC1, as suggested by the previous observation that MUC1 enhances cell surface expression of the Ca2+-selective channel, TRPV5, in cultured unpolarized cells. Using biotinylation of cell surface proteins, we asked whether MUC1 influences endocytosis of TRPV5 and another Ca2+-selective TRP channel, TRPV6, in cultured polarized epithelial cells. Our results indicate that MUC1 reduces endocytosis of both channels, enhancing cell surface expression. Further, we found that mice lacking MUC1 lose apical localization of TRPV5 and TRPV6 in the renal tubular and duodenal epithelium. Females, but not males, lacking MUC1 exhibit reduced blood Ca2+. However, mice lacking MUC1 exhibited no differences in basal urinary Ca excretion or Ca retention in response to PTH receptor signaling, suggesting compensation by transport mechanisms independent of TRPV5 and TRPV6. Finally, humans with autosomal dominant tubulointerstitial kidney disease due to frame-shift mutation of MUC1 (ADTKD-MUC1) exhibit reduced plasma Ca concentrations compared to control individuals with mutations in the gene encoding uromodulin (ADTKD-UMOD), consistent with MUC1 haploinsufficiency causing reduced bodily Ca2+. In summary, our results provide further insight into the role of MUC1 in Ca2+-selective TRP channel endocytosis and the overall effects on Ca concentrations.
Subject(s)
Calcium , Mucin-1 , TRPV Cation Channels , Animals , Female , Humans , Mice , Calcium/blood , Calcium/metabolism , Calcium/urine , Cell Membrane/metabolism , Cells, Cultured , Mucin-1/genetics , Mucin-1/metabolism , TRPV Cation Channels/metabolism , Epithelial Cells/metabolism , Sex Factors , Mutation , Protein Transport/geneticsABSTRACT
In situ monitoring of cell secretions and communications plays a fundamental role in screening of disease diagnostic biomarkers and drugs. Quantitative detection of cell secretions and monitoring of intercellular communication have been separately reported, which often rely on target labeling or complex pretreatment steps, inevitably causing damage to the target. Simultaneous in situ noninvasive detection of cell secretions and monitoring of intercellular communication are challenging and have never been reported. Herein, we smartly developed a portable device for in situ label-free monitoring of cell secretions and communications with fluorescence and ion-transport-based nanochannel electrochemistry. Based on the dual signal mode, a series of nonelectroactive secretions were sensitively and accurately quantified. The detection limits for VEGF, MUC1, and ATP were 3.84 pg/mL, 32.7 pg/mL, and 47.4 fM (3σ/S), which were 1/3.9, 1/1.1, and 1/41 of those of commercial ELISA kits, respectively. More interestingly, under the released secretions, the gradual opening of the nanochannel connected the two cells in the left and right chambers of the device; thus, the secretion mediated intercellular communication can be monitored. The proposed platform may provide a promising tool for understanding the mechanism of intercellular communication and discovering new therapeutic targets.
Subject(s)
Electrochemical Techniques , Humans , Electrochemical Techniques/instrumentation , Adenosine Triphosphate/analysis , Adenosine Triphosphate/metabolism , Mucin-1/analysis , Mucin-1/metabolism , Cell Communication , Vascular Endothelial Growth Factor A/analysis , Vascular Endothelial Growth Factor A/metabolism , Fluorescence , Limit of DetectionABSTRACT
Simultaneous profiling of redox-regulated markers at different cellular sublocations is of great significance for unraveling the upstream and downstream molecular mechanisms of oxidative stress in living cells. Herein, by synchronizing dual target-triggered DNA machineries in one nanoentity, we engineered a DNA walker-driven mass nanotag (MNT) assembly system (w-MNT-AS) that can be sequentially activated by oxidative stress-associated mucin 1 (MUC1) and apurinic/apyrimidinic endonuclease 1 (APE1) from plasma membrane to cytoplasm and induce recycled assembly of MNTs for multiplex detection of the two markers by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). In the working cascade, the sensing process governs the separate activation of w-MNT-AS by MUC1 and APE1 in diverse locations, while the assembly process contributes to the parallel amplification of the ion signal of the characteristic mass tags. In this manner, the differences between MCF-7, HeLa, HepG2, and L02 cells in membrane MUC1 expression and cytoplasmic APE1 activation were fully characterized. Furthermore, the oxidative stress level and dynamics caused by exogenous H2O2, doxorubicin, and simvastatin were comprehensively demonstrated by tracking the fate of the two markers across different cellular locations. The proposed w-MNT-AS coupled MS method provides an effective route to probe multiple functional molecules that lie at different locations while participating in the same cellular event, facilitating the mechanistic studies on cellular response to oxidative stress and other disease-related cellular processes.
Subject(s)
DNA-(Apurinic or Apyrimidinic Site) Lyase , DNA , Mucin-1 , Oxidative Stress , Humans , Mucin-1/metabolism , DNA/metabolism , DNA/chemistry , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Hydrogen Peroxide/metabolismABSTRACT
Bacteria inherently possess the capability of quorum sensing in response to the environment. In this work, we have proposed a strategy to confer bacteria with the ability to recognize targets with quorum-sensing behavior. Meanwhile, we have successfully achieved artificial control over the target-triggered aggregation of Escherichia coli (E. coli) by modifying the bacteria surface in a new way. Furthermore, by making use of green fluorescent protein (GFP) expressed by E. coli as the output signal, the aggregation of modified E. coli can be observed with the naked eye. Therefore, via the detection of the target, MUC1, an ovarian cancer biomarker, a simple and conveniently operated method to diagnose ovarian cancer is developed in this work. Experimental results show that the developed low-background and enzyme-free amplification method enables the highly sensitive detection of MUC1, achieving a remarkable limit of detection (LOD) of 5.47 fM and a linear detection range spanning from 1 pM to 50 nM and 50 nM to 100 nM, respectively. Clinical samples from healthy donors and patients can give distant assay results, showing great potential for clinical applications of this method.
Subject(s)
Escherichia coli , Mucin-1 , Ovarian Neoplasms , Ovarian Neoplasms/diagnosis , Escherichia coli/isolation & purification , Humans , Female , Mucin-1/analysis , Mucin-1/metabolism , Green Fluorescent Proteins/metabolism , Green Fluorescent Proteins/genetics , Limit of Detection , Biomarkers, Tumor/analysis , Biomarkers, Tumor/metabolism , Quorum Sensing , Biosensing Techniques/methodsABSTRACT
Accurate classification of tumor cells is of importance for cancer diagnosis and further therapy. In this study, we develop multimolecular marker-activated transmembrane DNA computing systems (MTD). Employing the cell membrane as a native gate, the MTD system enables direct signal output following simple spatial events of "transmembrane" and "in-cell target encounter", bypassing the need of multistep signal conversion. The MTD system comprises two intelligent nanorobots capable of independently sensing three molecular markers (MUC1, EpCAM, and miR-21), resulting in comprehensive analysis. Our AND-AND logic-gated system (MTDAND-AND) demonstrates exceptional specificity, allowing targeted release of drug-DNA specifically in MCF-7 cells. Furthermore, the transformed OR-AND logic-gated system (MTDOR-AND) exhibits broader adaptability, facilitating the release of drug-DNA in three positive cancer cell lines (MCF-7, HeLa, and HepG2). Importantly, MTDAND-AND and MTDOR-AND, while possessing distinct personalized therapeutic potential, share the ability of outputting three imaging signals without any intermediate conversion steps. This feature ensures precise classification cross diverse cells (MCF-7, HeLa, HepG2, and MCF-10A), even in mixed populations. This study provides a straightforward yet effective solution to augment the versatility and precision of DNA computing systems, advancing their potential applications in biomedical diagnostic and therapeutic research.
Subject(s)
DNA , Epithelial Cell Adhesion Molecule , MicroRNAs , Humans , Epithelial Cell Adhesion Molecule/metabolism , DNA/chemistry , MicroRNAs/analysis , MicroRNAs/metabolism , Mucin-1/metabolism , Mucin-1/analysis , Computers, Molecular , MCF-7 Cells , Biomarkers, Tumor/metabolism , Biomarkers, Tumor/analysis , Cell Membrane/metabolism , Cell Membrane/chemistry , Hep G2 CellsABSTRACT
Given that the corneal epithelium is situated on the outermost part of the eye, its functions can be influenced by external temperatures and chemical substances. This study aimed to elucidate the expression profile of chemosensory receptors in corneal epithelial cells and analyze their role in eye function regulation. A comprehensive analysis of 425 chemosensory receptors in human corneal epithelial cells-transformed (HCE-T) revealed the functional expression of TRPV4. The activation of TRPV4 in HCE-T cells significantly increased the expression of membrane-associated mucins MUC1, MUC4, and MUC16, which are crucial for stabilizing tear films, with efficacy comparable to the active components of dry eye medications. The present study suggests that TRPV4, which is activated by body temperature, regulates mucin expression and proposes it as a novel target for dry eye treatment.
Subject(s)
Epithelium, Corneal , Mucin-1 , Mucin-4 , Mucins , TRPV Cation Channels , Humans , CA-125 Antigen/metabolism , CA-125 Antigen/genetics , Epithelial Cells/metabolism , Epithelial Cells/cytology , Epithelium, Corneal/metabolism , Epithelium, Corneal/cytology , Membrane Proteins/metabolism , Membrane Proteins/genetics , Mucin-1/metabolism , Mucin-1/genetics , Mucin-4/metabolism , Mucin-4/genetics , Mucins/metabolism , Mucins/biosynthesis , TRPV Cation Channels/metabolism , TRPV Cation Channels/geneticsABSTRACT
BACKGROUND: Chimeric antigen receptor (CAR)-T cells have been used to treat blood cancers by producing a wide variety of cytokines. However, they are not effective in treating solid cancers and can cause severe side-effects, including cytokine release syndrome. TNFα is a tumoricidal cytokine, but it markedly increases the protein levels of cIAP1 and cIAP2, the members of inhibitor of apoptosis protein (IAP) family of E3 ubiquitin ligase that limits caspase-induced apoptosis. Degradation of IAP proteins by an IAP antagonist does not effectively kill cancer cells but enables TNFα to strongly induce cancer cell apoptosis. It would be a promising approach to treat cancers by targeted delivery of TNFα through an inactive adoptive cell in combination with an IAP antagonist. METHODS: Human dendritic cells (DCs) were engineered to express a single tumoricidal factor, TNFα, and a membrane-anchored Mucin1 antibody scFv, named Mucin 1 directed DCs expressing TNFα (M-DCsTNF). The efficacy of M-DCsTNF in recognizing and treating breast cancer was tested in vitro and in vivo. RESULTS: Mucin1 was highly expressed on the surface of a wide range of human breast cancer cell lines. M-DCsTNF directly associated with MDA-MB-231 cells in the bone of NSG mice. M-DCsTNF plus an IAP antagonist, SM-164, but neither alone, markedly induce MDA-MB-231 breast cancer cell apoptosis, which was blocked by TNF antibody. Importantly, M-DCsTNF combined with SM-164, but not SM-164 alone, inhibited the growth of patient-derived breast cancer in NSG mice. CONCLUSION: An adoptive cell targeting delivery of TNFα combined with an IAP antagonist is a novel effective approach to treat breast cancer and could be expanded to treat other solid cancers. Unlike CAR-T cell, this novel adoptive cell is not activated to produce a wide variety of cytokines, except for additional overexpressed TNF, and thus could avoid the severe side effects such as cytokine release syndrome.
Subject(s)
Dendritic Cells , Receptors, Chimeric Antigen , Tumor Necrosis Factor-alpha , Humans , Animals , Mice , Dendritic Cells/immunology , Dendritic Cells/metabolism , Female , Receptors, Chimeric Antigen/immunology , Tumor Necrosis Factor-alpha/metabolism , Mucin-1/immunology , Mucin-1/metabolism , Xenograft Model Antitumor Assays , Cell Line, Tumor , Inhibitor of Apoptosis Proteins/antagonists & inhibitors , Inhibitor of Apoptosis Proteins/metabolism , Immunotherapy, Adoptive/methods , Apoptosis , Breast Neoplasms/therapy , Breast Neoplasms/immunology , Immunotherapy/methods , Neoplasms/therapy , Neoplasms/immunology , Mice, SCIDABSTRACT
Lysosome-targeting chimera (LYTAC) links proteins of interest (POIs) with lysosome-targeting receptors (LTRs) to achieve membrane protein degradation, which is becoming a promising therapeutic modality. However, cancer cell-selective membrane protein degradation remains a big challenge considering expressions of POIs in both cancer cells and normal cells, as well as broad tissue distribution of LTRs. Here a logic-identification system is designed, termed Logic-TAC, based on cell membrane-guided DNA calculations to secure LYTAC selectively for cancer cells. Logic-TAC is designed as a duplex DNA structure, with both POI and LTR recognition regions sealed to avoid systematic toxicity during administration. MCF-7 and MCF-10A are chosen as sample cancer cell and normal cell respectively. As input 1 for logic-identification, membrane proteins EpCAM, which is highly expressed by MCF-7 but barely by MCF-10A, reacts with Logic-TAC to expose POI recognition region. As input 2 for logic-identification, Logic-TAC binds to POI, membrane protein MUC1, to expose LTR recognition region. As output, MUC1 is connected to LTR and degraded via lysosome pathway selectively for cancer cell MCF-7 with little side effect on normal cell MCF-10A. The logic-identification system also demonstrated satisfactory in vivo therapeutic results, indicating its promising potential in precise targeted therapy.
Subject(s)
Lysosomes , Membrane Proteins , Humans , Lysosomes/metabolism , Membrane Proteins/metabolism , MCF-7 Cells , Proteolysis , Animals , Mucin-1/metabolism , Logic , Cell Line, TumorABSTRACT
The expression pattern of MUC1-C in tumors is closely linked to tumor progression; however, its specific mechanism remains unclear. The expression of MUC1-C in cancer and adjacent normal tissues was detected using immunohistochemistry and Western blot. The IC50 of cells to gemcitabine was determined using the CCK8 assay. The effects of hypoxia and MUC1-C on the behavioral and metabolic characteristics of bladder cancer cells were investigated. Gene expression was assessed through Western blot and polymerase chain reaction. The relationship between the genes was analyzed by co-immunoprecipitation, immunofluorescence and Western blot. Finally, the role of the EGLN2 and NF-κB signaling pathways in the interaction between MUC1-C and hypoxia-inducible factor-1α (HIF-1α) was investigated. MUC1-C expression is significantly higher in bladder cancer tissues than in adjacent normal tissues, particularly in large-volume tumors, and is closely correlated with clinical features such as tumor grade. Tumor volume-mediated hypoxia resulted in increased expression of MUC1-C and HIF-1α in bladder cancer cells. Under stimulation of hypoxia, the inhibitory effect of EGLN2 on the NF-κB signaling pathway was weakened, allowing NF-κB to promote the positive feedback formation of MUC1-C and HIF-1α. Simultaneously, EGLN2-mediated degradation of HIF-1α was reduced. This ultimately led to elevated HIF-1α-mediated downstream gene expression, promoting increased glucose uptake and glycolysis, and ultimately resulting in heightened chemotherapy resistance and malignancy.
Subject(s)
Drug Resistance, Neoplasm , Hypoxia-Inducible Factor 1, alpha Subunit , Hypoxia-Inducible Factor-Proline Dioxygenases , Mucin-1 , Signal Transduction , Urinary Bladder Neoplasms , Female , Humans , Male , Middle Aged , Cell Hypoxia/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Gemcitabine , Gene Expression Regulation, Neoplastic/drug effects , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , Hypoxia-Inducible Factor-Proline Dioxygenases/genetics , Mucin-1/metabolism , Mucin-1/genetics , NF-kappa B/metabolism , NF-kappa B/genetics , Signal Transduction/drug effects , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/geneticsABSTRACT
BACKGROUND: Diabetic retinopathy (DR) is the leading cause of blinding eye disease among working adults and is primarily attributed to the excessive proliferation of microvessels, which leads to vitreous hemorrhage and retinal traction, thereby significantly impairing patient vision. NSUN2-mediated RNA m5C methylation is implicated in various diseases, and in this investigation, we focused on elucidating the impact of NSUN2 on the regulation of the expression of the downstream gene MUC1, specifically through RNA m5C methylation, on the progression of DR. METHOD: Utilizing Microarray analysis, we examined patient vitreous fluid to pinpoint potential therapeutic targets for DR. Differential expression of NSUN2 was validated through qRT-PCR, Western blot, and immunofluorescence in human tissue, animal tissue, and cell model of DR. The relationship between NSUN2 and DR was explored in vitro and in vivo through gene knockdown and overexpression. Various techniques, such as MeRIP-qPCR and dot blot, were applied to reveal the downstream targets and mechanism of action of NSUN2. RESULTS: The levels of both NSUN2 and RNA m5C methylation were significantly elevated in the DR model. Knockdown of NSUN2 mitigated DR lesion formation both in vitro and in vivo. Mechanistically, NSUN2 promoted MUC1 expression by binding to the RNA m5C reader ALYREF. Knockdown of ALYREF resulted in DR lesion alterations similar to those observed with NSUN2 knockdown. Moreover, MUC1 overexpression successfully reversed a series of DR alterations induced by NSUN2 silencing. CONCLUSIONS: NSUN2 regulates the expression of MUC1 through ALYREF-mediated RNA m5C methylation, thereby regulating the progression of DR and providing a new option for the treatment of DR in the future.
Subject(s)
Diabetic Retinopathy , Disease Progression , Methyltransferases , Mucin-1 , RNA Methylation , Animals , Humans , Male , Diabetic Retinopathy/metabolism , Diabetic Retinopathy/genetics , Diabetic Retinopathy/pathology , Gene Expression Regulation , Gene Knockdown Techniques , Methylation , Methyltransferases/metabolism , Methyltransferases/genetics , Mice, Inbred C57BL , Mucin-1/metabolism , Mucin-1/geneticsABSTRACT
In contrast to the decreasing trends in developed countries, the incidence and mortality rates of cervical squamous cell carcinoma in China have increased significantly. The screening and identification of reliable biomarkers and candidate drug targets for cervical squamous cell carcinoma are urgently needed to improve the survival rate and quality of life of patients. In this study, we demonstrated that the expression of MUC1 was greater in neoplastic tissues than in non-neoplastic tissues of the cervix, and cervical squamous cell carcinoma patients with high MUC1 expression had significantly worse overall survival than did those with low MUC1 expression, indicating its potential for early diagnosis of cervical squamous cell carcinoma. Next, we explored the regulatory mechanism of MUC1 in cervical squamous cell carcinoma. MUC1 could upregulate ITGA2 and ITGA3 expression via ERK phosphorylation, promoting the proliferation and metastasis of cervical cancer cells. Further knockdown of ITGA2 and ITGA3 significantly inhibited the tumorigenesis of cervical cancer cells. Moreover, we designed a combination drug regimen comprising MUC1-siRNA and a novel ERK inhibitor in vivo and found that the combination of these drugs achieved better results in animals with xenografts than did MUC1 alone. Overall, we discovered a novel regulatory pathway, MUC1/ERK/ITGA2/3, in cervical squamous cell carcinoma that may serve as a potential biomarker and therapeutic target in the future.
MUC1 is overexpressed in cervical squamous cell carcinoma. MUC1 regulates ERK phosphorylation, and subsequently upregulates ITGA2 and ITGA3 expression to promote tumorigenesis in cervical squamous cell carcinoma. A combination drug regimen targeting MUC1 and ERK achieved better results compared than MUC1 alone.
Subject(s)
Carcinoma, Squamous Cell , Cell Proliferation , Integrin alpha2 , Integrin alpha3 , Mucin-1 , Uterine Cervical Neoplasms , Humans , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/drug therapy , Female , Integrin alpha2/metabolism , Integrin alpha2/genetics , Animals , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/drug therapy , Mucin-1/metabolism , Mucin-1/genetics , Mice , Phosphorylation , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Xenograft Model Antitumor Assays , MAP Kinase Signaling System , Mice, Nude , Extracellular Signal-Regulated MAP Kinases/metabolismABSTRACT
The human gut symbiont Ruminococcus gnavus displays strain-specific repertoires of glycoside hydrolases (GHs) contributing to its spatial location in the gut. Sequence similarity network analysis identified strain-specific differences in blood-group endo-ß-1,4-galactosidase belonging to the GH98 family. We determined the substrate and linkage specificities of GH98 from R. gnavus ATCC 29149, RgGH98, against a range of defined oligosaccharides and glycoconjugates including mucin. We showed by HPAEC-PAD and LC-FD-MS/MS that RgGH98 is specific for blood group A tetrasaccharide type II (BgA II). Isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR confirmed RgGH98 affinity for blood group A over blood group B and H antigens. The molecular basis of RgGH98 strict specificity was further investigated using a combination of glycan microarrays, site-directed mutagenesis, and X-ray crystallography. The crystal structures of RgGH98 in complex with BgA trisaccharide (BgAtri) and of RgGH98 E411A with BgA II revealed a dedicated hydrogen network of residues, which were shown by site-directed mutagenesis to be critical to the recognition of the BgA epitope. We demonstrated experimentally that RgGH98 is part of an operon of 10 genes that is overexpresssed in vitro when R. gnavus ATCC 29149 is grown on mucin as sole carbon source as shown by RNAseq analysis and RT-qPCR confirmed RgGH98 expression on BgA II growth. Using MALDI-ToF MS, we showed that RgGH98 releases BgAtri from mucin and that pretreatment of mucin with RgGH98 confered R. gnavus E1 the ability to grow, by enabling the E1 strain to metabolise BgAtri and access the underlying mucin glycan chain. These data further support that the GH repertoire of R. gnavus strains enable them to colonise different nutritional niches in the human gut and has potential applications in diagnostic and therapeutics against infection.
Subject(s)
Clostridiales/metabolism , Mucin-1/metabolism , ABO Blood-Group System/immunology , Blood Group Antigens/immunology , Clostridiales/genetics , Clostridiales/physiology , Gastrointestinal Microbiome , Gastrointestinal Tract , Glycoside Hydrolases/metabolism , Humans , Mucins/metabolism , Oligosaccharides/metabolism , Polysaccharides/metabolism , Ruminococcus/genetics , Ruminococcus/metabolism , Substrate Specificity , Tandem Mass Spectrometry/methodsABSTRACT
Anti-cancer therapy is crucial in cancer prevention and anti-cancer, and thus, highly sensitive methods for detecting cancer biomarkers are essential for cancer early diagnosis. Herein, an electrochemical aptamer biosensor based on the CRISPR-Cas12a system was constructed for the detection of cancer tumor biomarker MUC1 mucin. The sensitivity was significantly prompted by enzyme-catalyzed signal amplification, and the selectivity was improved by the dual recognition of the aptamer to MUC1 and crRNA-Cas12a system to the aptamer. Glucose oxidase (GOD) was loaded on the surface of magnetic Fe3O4@Au (MGNP) via probe single-stranded DNA (pDNA) with the terminal modification of mercapto (-SH) to form GOD-pDNA/MGNP. The corresponding aptamer of MUC1 (MUC1 Apt) binds to its complementary ssDNA (cDNA) to form the activator Apt/cDNA, which is specifically recognized by crRNA-Cas12a and excites the trans-cleavage function of Cas12a, thus in turn trans-cleaves pDNA and detaches GOD from the magnetic particles. The magnetic beads were separated and transferred into a glucose solution, and the oxidation current of H2O2 produced by the catalytic reaction of GOD was measured on a Pt-modified magnetically-controlled glassy carbon electrode, resulting in an indirect determination of MUC1. The current change was linear with the logarithm of MUC1 concentration in the range from 1.0 × 10-17 g mL-1 to 1.0 × 10-10 g mL-1. The detection limit was as low as 7.01 × 10-18 g mL-1. The method was applied for the detection of MUC1 in medical samples.
Subject(s)
Aptamers, Nucleotide , Biomarkers, Tumor , Biosensing Techniques , Electrochemical Techniques , Glucose Oxidase , Mucin-1 , Humans , Aptamers, Nucleotide/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biomarkers, Tumor/genetics , Biosensing Techniques/methods , CRISPR-Associated Proteins/chemistry , CRISPR-Associated Proteins/metabolism , CRISPR-Cas Systems , DNA, Single-Stranded/chemistry , Electrochemical Techniques/methods , Endodeoxyribonucleases , Enzymes, Immobilized/chemistry , Glucose Oxidase/chemistry , Glucose Oxidase/metabolism , Gold/chemistry , Hydrogen Peroxide/chemistry , Limit of Detection , Magnetite Nanoparticles/chemistry , Mucin-1/metabolismABSTRACT
The transformation from normal to malignant phenotype in human cancers is associated with aberrant cell-surface glycosylation. It has frequently been reported that MUC1, the heavily glycosylated cell-surface mucin, is altered in both, expression and glycosylation pattern, in human carcinomas of the epithelium. The presence of incomplete or truncated glycan structures, often capped by sialic acid, commonly known as tumor-associated carbohydrate antigens (TACAs), play a key role in tumor initiation, progression, and metastasis. Accumulating evidence suggests that expression of TACAs is associated with tumor escape from immune defenses. In this report, we will give an overview of the oncogenic functions of MUC1 that are exerted through TACA interactions with endogenous carbohydrate-binding proteins (lectins). These interactions often lead to creation of a pro-tumor microenvironment, favoring tumor progression and metastasis, and tumor evasion. In addition, we will describe current efforts in the design of cancer vaccines with special emphasis on synthetic MUC1 glycopeptide vaccines. Analysis of the key factors that govern structure-based design of immunogenic MUC1 glycopeptide epitopes are described. The role of TACA type, position, and density on observed humoral and cellular immune responses is evaluated.
Subject(s)
Antigens, Neoplasm/immunology , Antigens, Tumor-Associated, Carbohydrate/immunology , Cancer Vaccines/immunology , Mucin-1/immunology , Polysaccharides/immunology , Vaccinology , Adjuvants, Immunologic , Animals , Antigens, Neoplasm/chemistry , Antigens, Tumor-Associated, Carbohydrate/chemistry , Antigens, Tumor-Associated, Carbohydrate/metabolism , Cancer Vaccines/adverse effects , Cancer Vaccines/therapeutic use , Cell Membrane/immunology , Cell Membrane/metabolism , Disease Progression , Humans , Immune Evasion , Immunotherapy , Lectins/metabolism , Mucin-1/chemistry , Mucin-1/metabolism , Neoplasm Metastasis , Neoplasms/immunology , Neoplasms/pathology , Neoplasms/therapy , Protein Binding , Vaccinology/methodsABSTRACT
BACKGROUND: Aberrant expression of MUC1 correlates with the progression of esophageal squamous cell carcinoma (ESCC), this study aimed to explore the effect of targeting MUC1 by Go-203 on malignant behavior of ESCC and the underlying mechanism. METHODS AND RESULTS: IHC was used to examine the expression of MUC1 and DNAJB6 in ESCC samples. qRT-PCR and western blotting were used to examine the expression of MUC1 and DNAJB6 in ESCC cell lines. CCK8, wound healing, and transwell assays were used to determine the effect of regulating MUC1/DNAJB6 on the proliferation, migration, and invasion of ESCC cells. The effect of overexpressing/targeting MUC1 on the activation of the AKT/HSF-1 pathway was determined by western blotting. A negative correlation was confirmed between the expression of DNAJB6 and MUC1 in ESCC tissue samples by IHC, and high expression of MUC1 and low expression of DNAJB6 correlated with lymph node metastasis in ESCC patients. Overexpressing MUC1 downregulated the expression of DNAJB6, promoted ESCC proliferation, invasion, migration and activated the AKT pathway, while targeting MUC1 suppressed proliferation, invasion, migration, and the AKT pathway and up-regulated DNAJB6 expression in vitro. Moreover, MUC1 increased the phosphorylation of HSF-1 via the AKT pathway, and inhibiting AKT-HSF-1 increased the expression of DNAJB6 in vitro. CONCLUSIONS: This study indicated that MUC1 could promote tumorigenesis and metastasis in ESCC by downregulating DNAJB6 expression through AKT-HSF-1 pathway.
Subject(s)
Esophageal Neoplasms , Esophageal Squamous Cell Carcinoma , Humans , Esophageal Squamous Cell Carcinoma/genetics , Esophageal Squamous Cell Carcinoma/metabolism , Esophageal Squamous Cell Carcinoma/pathology , Esophageal Neoplasms/metabolism , Lymphatic Metastasis , Proto-Oncogene Proteins c-akt/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Movement , Gene Expression Regulation, Neoplastic , Nerve Tissue Proteins/metabolism , Molecular Chaperones/metabolism , HSP40 Heat-Shock Proteins/genetics , HSP40 Heat-Shock Proteins/metabolism , Mucin-1/metabolismABSTRACT
GT-00AxIL15 is a novel interleukin-15-based immunocytokine targeting a tumor-specific, glycosylated epitope of MUC1 (TA-MUC1). We characterized mode of action, pharmacokinetic (PK) and pharmacodynamic (PD) properties and investigated the relevance of TA-MUC1 binding for the concept of delivering IL-15 to solid tumors. In vitro pharmacology was analyzed in binding and cell-based assays. The in vivo PK profile and IL-15-mediated PD effects of GT-00AxIL15 were investigated in tumor-free mice. Tumor accumulation, immune infiltration and anti-tumor activity were assessed in TA-MUC1+ syngeneic and xenogeneic murine tumor models. GT-00AxIL15 was shown to specifically bind TA-MUC1 on tumor cells via its mAb moiety, to IL-15 receptors on immune cells via its IL-15 fusion modules and to FcγRs via its functional Fc-part. In vitro, NK, NKT and CD8+ T cells were activated and proliferated, leading to anti-tumor cytotoxicity and synergism with antibody-dependent cellular cytotoxicity (ADCC)-mediating mAbs. In vivo, GT-00AxIL15 exhibited favorable PK characteristics with a serum half-life of 13 days and specifically accumulated in TA-MUC1+ tumors. In the tumor microenvironment, GT-00AxIL15 induced robust immune activation and expansion and mediated anti-metastatic and anti-tumor effects in syngeneic and xenograft tumor models. These results support the rationale to improve PK and anti-tumor efficacy of IL-15 by increasing local concentrations at the tumor site via conjugation to a TA-MUC1 binding mAb. The tumor-selective expression pattern of TA-MUC1, powerful immune activation and anti-tumor cytotoxicity, long serum half-life and tumor targeting properties, render GT-00AxIL15 a promising candidate for treatment of solid tumors with high medical need, e.g., ovarian, lung and breast cancer.